Classifying Your Thyroid FNA Specimens

Using Bethesda Terminology: Use of

Adjunct Molecular Reflex Testing
Constantine Theoharis, MD
Assistant Professor
David Chhieng, MD, MBA, MSHI
Professor
Director of Cytology
Department of Pathology
Yale University
New Haven CT

Overall Objectives

To apply the Bethesda Thyroid FNA Classification

System in the evaluation of thyroid FNA.

To utilize molecular testing as an adjunctive test in

thyroid FNA.

To advise clinicians on the implications of the each

diagnostic category of the Bethesda Classification

system and the results of molecular testing.

Overview

Usage and clinical implications of the Bethesda Thyroid

Fine Needle Aspiration Classification System

Indications and implications of molecular testing in thyroid

FNA

The Utilization of the Bethesda

System & BRAF Molecular
Adjunct Testing on Thyroid
Cytologic Samples
Part I of II
Constantine Theoharis, MD
Assistant Professor
Department of Pathology
Yale School of Medicine
ASCP 2011

Objectives

Review the Bethesda

Thyroid FNA Classification

System

Describe its utilization at

Yale & the questions it

raises

Cellularity

Posit molecular testing as

prequel to part 2

Challenge our view of the

Architecture

Cytology

modern
cytopathologist/surgical
pathologist
The Art of Cytopathology
5

Background

Thyroid Cancer

Most common endocrine

malignancy

Incidence increasing

Problematic clinically

Nodules

~7% of the population

Only 5% are malignant

Thyroid FNA (TFNA) =

the keystone modality

Diagnostic test*

Screening test *

*more in a moment

But TFNA is Compromised

60% benign

10% malignant

20% equivocal

10% nondiagnostic/unsatisfactory

Lack of Uniformity

Terminology

Criteria

Clinical Implications

Bethesda Classification

Resolutions?

Molecular Diagnostics

Not expressly advocated in
the Bethesda System
6

The Bethesda System

1.

2.

Nondiagnostic or
Unsatisfactory

Cyst fluid only

Virtually acellular specimen

Obscuring factors

Specify if Hrthle cell type

5. Suspicious for malignancy

Benign

3.

4. Follicular neoplasm or
suspicious for a follicular
neoplasm

Benign follicular nodule

e.g. adenomatoid
nodule, colloid nodule
Lymphocytic thyroiditis

Atypia of undetermined
significance

6. Positive for malignancy

PTC

Medullary carcinoma

Anaplastic carcinoma

Lymphoma

Metastatic neoplasm

Other

Ali and Cibas (Ed): The Bethesda System for Reporting Thyroid Cytopathology. Springer. 2009

Audience Response
How many thyroid FNAs do you see at your
institution?

Answer choice #1: 1-100 cases/year.

Answer choice #2: 101-500 cases/year.

Answer choice #3: 501-1000 cases/year.

Answer choice #4: 1001-3000 cases/year.

Answer choice #5: >3000 cases/year.

Yale Endocrine Cytopathology

and Surgical Pathology

Endocrine Surgery

Referral Center

Over 3,200 Thyroid

FNAs annually

Representing 2,500

patients seen in
clinic per year

Approximately 400

operations
performed annually
Yale Smilow Cancer Center
9

Audience Response
Do you utilize the Bethesda Thyroid FNA
Classification at your institution?

Answer choice #1: No. We dont have a

classification system per se.

Answer choice #2: No. We have our own

departmental system.

Answer choice #3: Yes. We use TBS as it has been

described in the literature.

Answer choice #4: Yes, however, weve modified it

somewhat to fit our needs.

The Bethesda System (Yale Version)

1.

Nondiagnostic/ Unsatisfactory

2.

Benign

3.

Insufficient cellularity
Poor preservation
Obscuring factors

Benign mixed macro/microfollicular hyperplastic nodule

i.e. goiter
Lymphocytic thyroiditis/
Hashimoto thyroiditis
Cyst Contents or Colloid
Nodule-if USG matches

Indeterminate [AUS/FLUS]

Low cellularity with

predominance of microfollicles
and absence of colloid
Atypical nuclear features

4.

Follicular neoplasm

Microfollicular pattern
S/O Follicular Variant of PTC

5.

Hrthle cell neoplasm

6.

Suspicious for malignancy

7.

Papillary Thyroid Carcinoma

Medullary carcinoma
Anaplastic carcinoma
Lymphoma
Metastatic malignancy

Positive for malignancy

Papillary Thyroid Carcinoma

Medullary carcinoma
Anaplastic carcinoma
Lymphoma
Metastatic malignancy

Theoharis et al. Thyroid 2009;19:1215

11

Is Thyroid FNA a Diagnostic or a

Screening Test?

Diagnostic

Screening

Positive for PTC/MTC/ATC

Follicular Neoplasm

Suspicious for

Hrthle Cell Neoplasm

Negative for Malignancy

AUS/Indeterminate?

AUS/Indeterminate?

Architecture

Group Cell Features

Single Cell Features

Cytology

Nucleus v.s. Cytoplasm

Architecture

Group Cell Features

Single Cell Features

Cytology

Nucleus v.s. Cytoplasm?
12

Cyto-Method Differs from Histology

Cellularity

Follicular Cells

Adequacy

6 groups

10-15 cells/group

Colloid

Not needed

But useful

Lymphs /Macs/ Others?

Architecture

Group Cell Features

Single Cell Features

Cytology

Nucleus vs. Cytoplasm

13

Negative
USG: Hyperechoic nodule(s), capsular calcifications, bordering vesselsnon-specific

15

Papillary Thyroid Carcinoma

Ultrasound

Hypoechoic

Microcalcifications

Increased Vascularity

Cellularity

Follicular Cells!

Architecture

+/- Papillae, sheets,

caps

Cytology

Irregular Nuclear

Membranes & Grooves

Intra-Nuclear
Cytoplasmic
Invaginations (INCIs)

16

Papillary Thyroid Carcinoma Papillae

17

Papillary Thyroid Carcinoma Inclusion (INCI)

18

Psammoma Body (Calcospherite)

19

Papillary Thyroid CarcinomaPapillae

20

Papillary Thyroid Carcinoma

Inclusion (INCI)

Does a classic positive PTC need molecular testing? BRAF? For Dx? for Pgx?

21

PTC Papillae & Psammoma

22

PTC with Oncocytic Features

23

Papillary Thyroid Carcinoma

to LN

24

Papillary Thyroid Carcinoma

to LN

Often, few if any lymphocytes are present

25

Follicular & Hrthle Cell

Thyroid Neoplasms

Architecture

Microfollicles

NO Papillae

Capsule & Vessel?

Cytoplasm

Amount v.s. Nucleus

Nuclei

USG: Isoechoic nodule, no calcifications, no

central vascularitynon-specific findings

Smooth

Enlarged

+/- Nucleoli

+/- Scant Colloid

NO INCIs

26

Follicular Adenoma Capsule

27

Follicular Thyroid Carcinoma

CAPSULAR INVASION

VASCULAR INVASION

Architecture

Microfollicles

NO Papillae

Nuclei

Smooth

Enlarged

+/- Nucleoli

+/- Scant Colloid

NO INCIs
28

FTC Capsular Mushrooming

29

Follicular Thyroid Carcinoma

30

Follicular Neoplasm >

Follicular Adenoma

31

Follicular Neoplasm >

Follicular Carcinoma

32

Hrthle Cell Carcinoma

Vascular Invasion

33

Hrthle Cell Neoplasm

34

Hrthle Cell Neoplasm >

Hrthle Cell Carcinoma
Normal sized cells

Massive cell with atypia

Rarely, the degree of atypia is so great

35

Indeterminate/FLUS/AUS

For specimens that contain cells (follicular, lymphoid, or

other) with architectural and/or nuclear atypia

insufficient to be classified as suspicious for a follicular
neoplasm, suspicious for malignancy, or malignancy

Criteria

Borderline cellularity with predominance of follicular cells

and absence/scant colloid

Borderline cellularity with predominance of Hrthle cells
Focal nuclear atypia s/o PTC (nuclear enlargement with
pale chromatin, nuclear grooves) particularly in patients
with lymphocytic thyroiditis or cystic changes
Atypical lymphoid population
Atypia with obscuring factors and/or air drying artifacts
36

AUS/FLUS/Indeterminate
(Yale Version)

Architectural Atypia

Low Cellularity

Microfollicles

Absent/Scant Colloid

Incipient Changes of

Neoplasia?

37

Indeterminate-Architectural Atypia

Follicular Adenoma with

intact capsule on resection

38

AUS/FLUS/Indeterminate
(Yale Version)

Nuclear Atypia

Elongation/Enlargement

Nuclear Membrane

Irregularities/Grooves

Rare possible(?)
pseudoinclusions

Incipient Changes of

Neoplasia?

Indeterminate-Nuclear Atypia
39

Mixed Classic and Follicular

variant PTC on resecton

40

Indeterminate/FLUS/AUS

At our institution, the term indeterminate, corresponds

to the NCI 2007 guidelines category Follicular cells of

undetermined significance. Lesions designated as such
my benefit from re-aspiration in the appropriate clinical
context.

Repeat FNA in 3- 6 months

~20% of nodules are repeatedly diagnosed as

indeterminate

Surgery indicated if worrisome clinical and/or US findings

Can we do better?

Reflex BRAF mutational analysis?*
*more on this coming in part 2!

41


GOAL:

Maintain high PPV of
malignancy on f/u but
w/o compromising
sensitivity

Suspicious for
malignancy

Quantity

Quality

The Need for Compromise

USUAL SUSPECTS:

Suspicious for PTC

Most common

Lobectomy frozen
section (completion
subsequently)

Suspicious MTC, ATC, NHL

Compromised:

Quantity

Quality

Can we do better?

Reflex BRAF mutational

analysis?*

*more on this coming in part 2!

42

Suspicious for malignancy

Papillary group

Nuclear atypia

Overstained, obscuring blood, low cellularity, compromise dsample

43

Whats the difference?

Is it Atypical?

Is it Suspicious

Rule of Thumb:

Rule of Thumb:

Im not certain its

Im not certain its

negative

positive

You the cytopathologist

You the cytopathologist

are communicating:

are communicating:

Dont lose this patient to

Consider lobectomy

follow-up

Malignant risk should be

low: <30%

based upon this sample

Malignant risk should be

high: >60%

44

Medullary Thyroid Carcinoma

Cellularity

Cellular

Architecture

SINGLE Cells

Cytology

Spindled

Plasmacytoid

Nuclei

Smooth

Salt & Pepper

Small nucleoli

+/- INCIs

Ancillary Test?

Calcitonin

45

Medullary Thyroid Carcinoma

46

Medullary Thyroid Carcinoma

47

Anaplastic Thyroid Carcinoma

History

Older patient

Rapid, recent growth

Gross

Hemorrhage

Necrosis

Architecture

SINGLE/Cellular
Giant/Spindled
Pleomorphic
PMNs

Nuclei

Dark/Irregular

Mitoses

INCIs

48

ATC Immunostains

Thyroglobulin

TTF-1

Calcitonin

CK-7

49

Anaplastic Thyroid
Carcinoma

50

Anaplastic Thyroid
Carcinoma

51

Non-Hodgkin Lymphoma

Large B-Cell

Hashimoto association

Architecture

Monotypic/Cellular

Cytoplasmic vacuoles

Nuclei

Immature chromatin

Multi-Nucleoli

52

Thyroid NHL

Liquid Monolayer pseudo-groups

53

Thyroid NHL +CD45

54

Distribution of cytologic categories

Cytologic Category

By Nodules
2008

By Patients
2008

Expected frequency

Unsatisfactory

357 (11.7%)

230 (9.0%)

10% to 15%

Benign/Negative for
Malignancy

2368 (78.0%)

1799 (72.8%)

70% to 80%

Indeterminate/Atypia of
Undetermined
Significance

95 (3.0%)

89 (3.6%)

3% to 18%

Follicular /Hrthle Cell

Neoplasm

176 (5.8%)

166 (6.7%)

5% to 8%

Suspicious for
Malignancy*

43 (1.4%)

39 (1.6%)

2.5% to 8%

Malignancy*

168 (5.5%)

145 (5.9%)

4% to 8%

3207

2468

Total

* Majority of them were PTC

Modified from Theoharis et al. Thyroid

2009;19:1215

55

Comparison before and after TBS

Cytologic Category

By Nodules
2008

By Patients
2008

By Nodules
2007

By Patients
2007

Unsatisfactory*

357 (11.7%)

230 (9.0%)

293 (14.4%)

197 (12%)

Benign/Negative for
Malignancy*

2368 (78.0%)

1799 (72.8%)

1361 (66.9%)

1053 (65.9%)

Indeterminate/AUS

95 (3.0%)

89 (3.6%)

Susp FN 48
(2.3%)

Susp FN 45
(2.8%)

Follicular /Hrthle Cell

Neoplasm*

176 (5.8%)

166 (6.7%)

174 (8.3%)

156 (9.8%)

Suspicious for
Malignancy

43 (1.4%)

39 (1.6%)

29 (1.4%)

26 (2%)

Malignancy

168 (5.5%)

145 (5.9%)

119 (6%)

112 (7%)

Total

3207

2468

2035

1596

* Differences were statistically significant

Theoharis et al. USCAP 2010

56

Cytologic-Histologic Correlation
MNG/HT

FA

CA

Total

25

Benign/Negative for
Malignancy (0.3%)
Indeterminate (30%)

61

13

8*

82

13

27

Follicular / Hrthle Cell

Neoplasm (61%)
Suspicious for
Malignancy (77%)
Malignancy (77%)

33

34

35**

102

26

30

112

112

Total

112

64

202

378

Cytologic category
(% surgery)
Unsatisfactory (11%)

*The false negatives were micro PTC ( 1cm), not initially sampled by FNA
** included both follicular CA and FV PTC
57

Operating Characteristic

Sensitivity
Specificity
Positive predictive
value
Negative predictive
value

As a Screening test
for NEOPLASM
NA
68%

As a Diagnostic test
for MALIGANCY
NA
93%

NA

NA

83%

91%

Theoharis et al. Thyroid 2009;19:1215

58

Risk of malignancy per Dx

Diagnostic Category

Incidence of
malignancy at Yale

NCI recommended
rate of malignancy

Benign/Negative for
Malignancy

10%* (0.3%)

0%-3%

Indeterminate

30%* (14%)

5%-15%

Follicular /Hrthle Cell

Neoplasm

33%

20%-30%

Suspicious for Malignancy

87%

60%-75%

100%

97%-99%

Malignancy

* Only a selected subset of patients underwent surgery

Modified from Theoharis et al. Thyroid
2009;19:1215

59

Indeterminate/FLUS/AUS

171 nodules diagnosed as indeterminate/FLUS/AUS

between Jan 2008 to Jun 2009;

Accounting for 2.8% of all cases
Category

Number of
cases

Case with Follow-Up

(Surgery/Repeat FNA)

Malignant
Follow-Up

Low cellularity/
microfollicular
pattern

104 (61%)

59(59%/41%)

7%

Nuclear atypia

67 (39%)

45(73%/27%)

56%

171

104 (65%/35%)

20%

Total

60

Adeniran et al USCAP 2010

Problems with equivocation

On Re-FNA

<10% of Indeterminates
were re-dxd as
Indeterminate

Majority are PTC; most

classified as having nuclear

atypia cytologically

Re-FNA benign?

Hyperplastic nodules in
both groups with Hashimoto
thyroiditis more prevalent in
the 2nd group

Suspicious Category less

problematic (87% CA risk)

Adjunct testing?

Immunostaining?

Molecular testing?

61

Molecular Diagnostics?
primum non nocere*

* From the
Greek:
,

MAPK SIGNALING PATHWAY

62

Audience Response
Do you utilize molecular testing on thyroid FNAs at
your institution?

Answer choice #1: No.

Answer choice #2: BRAF only.

Answer choice #3: BRAF, RET only.

Answer choice #4: BRAF, RAS only.

Answer choice #5: Panel of BRAF, RET, RAS, PAX

etc.

A Possible Guideline to Molecular

Thyroid FNA Testing

Theoharis C, Hui P. Surgery of the Thyroid and Parathyroid Glands

2nd Ed. (in press)

64

Histology
Cytology

Gross
Pathology
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Diagnostics
Electronic Data Layer
Practice

Immuno &
Chem Stains
Electron
Microscopy

Flow
Cytometry

Management

Image
Analysis

Knowledge
Databases

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Arrays

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Genomics

Pathologist

Proteomics
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Decision

Pharmacogenomics

Support

OUTCOME

Epidemiology

Medical
Literature

Diagnostic
Imaging

Medical
Record

Clinical
Laboratory

Dx
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Options
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Techniques

Treating
Clinician(s)
Rx
Patient

Patient
Health
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Environment

Pathologist/Cytopathologist as
Diagnostic Specialist
Pathology develops and
adopts tools to leverage
data from emerging
technologies
We become diagnostic
consultants, providing
outcomes based treatment
recommendations to
treating clinicians
We become the
Department of Diagnostic
Medicine

Sinard JH, Practical Pathology Informatics,

used with permission

65

Summary

The Bethesda 6-tier classification system

Conveys different levels of risk of malignancy

Excellent screening test for follicular/Hrthle cell

neoplasm

Superb diagnostic test for identifying PTC with a
specificity of 93%

Sub-classifying indeterminate category into 2

descriptive groups conveys different levels of risk

Molecular Testing may have a role especially in

equivocal cases

Part 2 follows shortly

Thank you!
66

Acknowledgements

Cytopathologists

David Chhieng

Adebowale Adeniran

Diane Kowalski

Malini Harigopal

Guoping Cai

Angelique Levi

Surgical Pathologist

Manju Prasad

Molecular Pathologist

Pei Hui

Surgeons

Robert Udelsman

Sanziana Roman

Julie Ann Sosa

Tobias Carling

Radiologist

Lynwood Hammers

67

Molecular Testing in Thyroid FNA

David Chhieng, MD, MBA, MSHI

Professor
Director of Cytology
Department of Pathology
Yale University
New Haven CT

Objectives

To apply the Bethesda Thyroid FNA Classification
System in the evaluation of thyroid FNA.

To utilize molecular testing as an adjunctive test in

thyroid FNA.

To advise clinicians on the implications of the each

diagnostic category of the Bethesda Classification
system and the results of molecular testing.

Overview

Introduction

BRAF testing as a diagnostic marker

RAS mutational analysis

Use of a molecular panel

BRAF testing as a prognostic marker

Adjunctive Testing

Immunocytochemistry

Flow cytometry

Lymphocytic thyroiditis vs lymphoma

Molecular testing

Immunocytochemistry

Primary vs secondary

TTF-1 and thyroglobulin

Medullary carcinoma

Calcitonin, chromogranin, synaptophysin, and mCEA

PTC vs other follicular-derived lesions

CK 19, HBME-1, Galectin-3

None specific enough

False positive staining in Non-neoplastic lesions such
as lymphocytic thyroiditis and post FNA reactive foci

An example of medullary carcinoma staining positive

for calcitonin (ThinPrep preparation)

Immunohistochemistry for cytokeratin 19 in a fine needle aspirate of papillary

carcinoma (Giemsa preparation with immunohistochemistry performed after
removal of the coverslip).

Anderson C E , McLaren K M J Clin Pathol 2003;56:401-405

Expression of various makers in

thyroid lesions
Diagnosis

Galactin -3

HBME 1

CK 19

92%

87%

72%

PTC (n=67)

94%

85%

72%

Follicular (n=6)

66%

50%

50%

Hurthle cell (n=8)

88%

13%

50%

Anaplastic (n=4)

100%

0%

25%

Adenoma (n=21)

10%

10%

5%

Non neoplastic thyroid (n=102)

17%

7%

13%

Nodular goiter (n=29)

55%

24%

31%

Thyrotoxicosis (n=14)

7%

0%

0%

Normal (n=59)

0%

0%

7%

Carcinoma (n=85)

Prasad et al. Mod Pathol. 2005:14:169.

Molecular Testing

Audience Response
What molecular testing does your laboratory offer for
thyroid FNA?

1. None

2. BRAF only

3. BRAF and Ras

4. A panel of markers (i.e. BRAF, Ras, RET/PTC, PAX8PPR

)

Revised Management Guidelines for

Patients with Thyroid Nodules and
Differentiated Thyroid Cancer
American Thyroid Association, 2009

MAPK Signaling pathway

>70% of PTC found
to have mutations
involving one of
the genes in the
MAPK signaling
pathway

Especially RET/PTC,
RAS, and BRAF

Usually mutually
exclusive

Nikiforov Y. Mod Path. 2008:2: S37

Other mutations

Follicular carcinoma

RAS mutation

PAX8-PPAR rearrangement

Medullary carcinoma

RET point mutation

Molecular alterationsfrequency
Abnormality

FA

F CA

PTC

Comments

BRAF

--

--

RET/PTC

--

--

RAS

20-40%

40-50%

10-20% 5% in goiter
FV PTC
Distant > LN metastasis

PAX8-PPAR
(t2,3)(q13;p25)

2-10%

20-40%

5-10%

40-60% Classic and Tall cell

Extranodal extension
and LN metastatsis
20%

Classic
Young onset and post
radiation
Type 1 (70%)
Type 3 (30%)

FV PTC
Younger age
Aggressive behavior

Modified from Gillian CP. ANZ J Surg. 2010; 80: 33

BRAF

Found in ~45% (40-60%) of PTC

Virtually all point mutations result in a valine-toglutamate at residue 600 (V600E)

Highly prevalent in classical and tall cell variant

Rare in follicular variant

Specificity of BRAF Detection in Thyroid FNA

Nature of studies

No of
samples

BRAF
positive

Final diagnosis in
BRAF-positive samples

Prospectives

1814

159

100%

Retrospectives

685

291

100%

Research FNA of
surgically
removed thyroid

267

131

99.2%*

Total

2766

581

99.8%

The false positive is hyperplastic nodule with atypical nodular hyperplasia

Nikiforova & Nikiforov. Thyroid. 2009;19:1351

BRAF

Using SSCP, BRAF detected in 76% (28/37)
classical PTC (Yale data)

Compared to direct sequencing, BRAF detected
in 65% (24/37)

SSCP had an analytical sensitivity of 5% tumor cells
for a definite identification of BRAF

Implemented reflex BRAF testing for equivocal

and positive thyroid FNA since Sept 09

A total of 157 thyroid FNAs tested between Sept

2009 and Nov 2010

156 (99.4%) had sufficient DNA for BRAF testing

Demographic and characteristics of

patients and thyroid nodules

^10 patients had two nodules biopsied

* the case with insufficient DNA was excluded

# According to nodules

^10 patients had two nodules biopsied

* the case with insufficient DNA was excluded
# According to nodules

Adeniran et al. Thyroid 2011; 21:717

Histologic follow up results of thyroid

nodules with equivocal cytologic diagnosis

Over 50% of suspicious cases found to be follicular variant of PTC

Adeniran et al. Thyroid 2011; 21:717

Tissue follow up of thyroid nodules with

positive cytologic diagnosis according to
BRAF status

Adeniran et al. Thyroid 2011; 21:717

Histologic follow up with indeterminate

FNA and BRAF testing
Cytologic
diagnosis
(n=84)

BRAF
results

Indeterminate
w/ MF pattern
(INa)
Indeterminate
w/ nuclear
atypia (INb)

Surgical Follow up

Total

PTC

Benign

Positive
(n=0)

Negative
(n=28)

2*

10

Positive
(n=19)

16

16

Negative
(n=36)

9*

13

22

P value

< 0.001

These cases were follicular variant and diffuse sclerosing variant PTC

Adenirian et al. Acta Cytologica. In press

Performance of BRAF testing in

indeterminate FNA
Sensitivity

Specificity

PPV

NPV

59%

100%

100%

66%

~ 20% increase in positive identification of PTC

Adenirian et al. Acta Cytologica. In press

Algorithm for managing patients with

thyroid FNA and BRAF testing

Ras Mutations

Point mutations found in many human cancers and in most
types of thyroid tumors

K-RAS, H-RAS, N-RAS genes may be involved

Hot spots - codons 12, 13 and 61

N-RAS codon 61 mutations most common in thyroid tumors

Mechanism of RAS Activation by

Point Mutation

RAS

GDP

Inactive

Mutations
codons
12/13 in exon 1
affecting GTP
binding domains

Active
RAS

GTP

Downstream
Effectors

61 in exon 2
affecting GTPase
domain

Molecular Pathways Activated by RAS

RET
P

SHC
Y1062

PI3K
AKT
BAD

P70S6K

FRS2

GRB2

PLC

PKC
c-Jun, Fos ,
c- Myc , Elk-1

RAS

B-RAF
MEK

DAG

BCL

Apoptosis

SOS

Ral /Cdc42

MEKK1

Rac

JNK

Rho

ERK

Incidences of Ras Mutations in Thyroid

Lesions
Thyroid Lesions

Incidence of Ras
Mutations

Follicular Carcinoma

40-50%

Papillary Carcinoma

10-20%*

Follicular Adenoma

20-40%

Goiter (Adenomatous Hyperplasia)

0-3%

Almost exclusively in follicular variant

Liu et al. Thyroid 2004: 14: 616
Nikiforvoa and Nikiforvoa. Thyroid 2009: 19: 1351

RAS mutations and

indeterminate FNA

64 Indeterminate thyroid FNAs (including FLUS, FN,
and suspicious) with positive RAS mutation and
surgical follow up

Results of surgical follow up

Malignant

Neoplastic
Non-neoplastic

84%
PTC

52

Follicular carcinoma

Follicular Adenoma

11

16%

0%

Nikiforov et el. JCEM. 2009;94:2092

Our Approach

Perform RAS mutation in addition to BRAF mutation analysis for all
indeterminate cases

Positive BRAF
(regardless of
RAS mutation)

Indeterminate

Total
thyroidectomy

Positive
RAS/Negative
BRAF

Lobectomy

Negative BRAF +
Negative RAS

Repeat FNA

Our Approach

Not recommended for cases with the diagnosis of
Follicular Neoplasm

Up to 20% of RAS positive cases are Follicular
Adenoma and occasionally Adenomatous
Hyperplasia

Current approach is lobectomy +/- intraoperative
consultationNo significant impact on patient
management

Alternate approach to molecular testing

in thyroid FNA

Performing a panel of mutations on ALL thyroid FNAs

The panel includes

BRAF (V600E)
RAS (k-,n-, & h-)
RET/PTC rearrangement (RET/PTC 1 & RET/PTC 3)
PAX8-PPR

Performance of Molecular
testing using a panel
Reference

Number of FNA
Samples

Specimens
types

Number of mutations
identified

470

All types

32 (7%)

400

All types

50 (13%)

285

Only those with

surgical FU

67 (29%)

432

All types

61 (14%)

1.
2.
3.
4.

Nikiforov et el. JCEM. 2009;94:2092.

Moses et al. WJ Surg. 2010;34:2589.
Cantara et al. JCEM. 2010;95:1365.
Mathur et al. Surgery. 2010; 148:1170.

Diagnostic
category

Numbers

Positive

79

Suspicious

Indeterminate

Negative

Non-diangostic

Total

1.
2.
3.

81

115

99

53

427

Nikiforov et el. JCEM. 2009;94:2092.

Moses et al. WJ Surg. 2010;34:2589.
Cantara et al. JCEM. 2010;95:1365.

Mutation status

Follow up
Malignant

Follicular
Adenoma

Negative

Positive

26

26

Negative

53

53

Positive

52

50

Negative

29

10

15

Positive

22

21

Negative

93

29

29

35

Positive

13

Negative

86

10

71

Positive

14

12

Negative

39

11

24

Positive

127
(30%)

118
(93%)

7
(6%)

2
(1%)

Negative

300
(70%)

101
(34%)

54
(18%)

145
(48%)

Performance of Molecular
testing using a panel
Reference

Sensitivity

Specificity

PPV

NPV

62%

100%

97%

95%

38%

65%

42%

65%

80%

100%

100%

90%

1. Nikiforov et el. JCEM. 2009;94:2092.

2. Moses et al. WJ Surg. 2010;34:2589.
3. Cantara et al. JCEM. 2010;95:1365.

Prognostic Molecular
Markers

BRAF and Prognosis

Correlate with aggressive tumor characteristic

Extrathyroidal extension
Nodal metastases
Resistant to radioiodine
Tumor recurrence

BRAF: Invasion and LNs

BRAF: Recurrence

Kaplan-Meier estimate of recurrence-free probability of PTC in patients with or without BRAF

mutation.
A, Analysis of a multicenter series consisting of 219 cases, mainly Caucasian patients. Log-rank
test: 2 4.0, P 0.04. [Xing et al., 2005 (83).]
B, Analysis of a Korean series consisting of 203 patients. Log-rank test: 2 4.60, P 0.037.
[Kim et al., 2006 (71), with permission from WileyBlackwell.]

BRAF: Higher Stage Disease

BRAF is an
Independent
Prognostic Factor
associated with
Worse Disease and
Poorer Outcomes
Xing, M: BRAF Mutation
in Thyroid Cancer

BRAF mutation analysis and FNA

BRAF +ve

BRAF ve

Follicular variant

7%

51%

p<0.05

Classic

22%

11%

p<0.05

Tall cell variant

30%

6%

p<0.05

Extrathyroidal extension

56%

15%

p<0.05

Mean size (cm)

1.9

2.3

p<0.05

LN involvement (Level VI)

47%

19%

p<0.05

Histologic subtype

Yip et al. Surgery 2009; 146: 1215

Pathologic features of 60 papillary thyroid

carcinomas

Adeniran et al. Thyroid 2011; 21:717

Histologic Features of Papillary Thyroid

Carcinoma with and without BRAF Mutation
Morphologic features
Tumor Capsule (intact or
infiltrated)
Infiltrative Growth

BRAF mutation
Positive
12 (35%)1

BRAF mutation
Negative
15 (68%)2

P Value

32 (94%)

13 (59%)

0.002

33 (97%)

15 (68.1%)

<0.001

22 (65%)

6 (27%)

0.01

0.02

Stromal fibrosis, sclerosis or

desmoplasia
Plump tumor cells with
moderate amount of
eosinophilic cytoplasm
Classic fully developed nuclear
features
Lymphovascular invasion

33 (97%)

5 (22.7%)

0.0001

4 (11.7%)

4 (18%)

NS

Psammoma bodies

17 (50%)

5 (22.7)

0.05

Stromal calcifications

24 (70%)

10 (45%)

0.09

Cystic change

8 (23.5%)

3 (13.6%)

NS

Extrathyroidal Extension

4 (11.7%)

4 (18%)

NS

Finklestein et al. Presented at the 2011 USCAP meeting

Potential impact of BRAF status

on patient management

Initial surgical management

Total thyroidectomy

Possible central lymph node dissection

Initial radioiodine treatment

Higher does of radioactive iodine

Audience Response
What kind of specimen does your laboratory used
for molecular testing for thyroid FNA?
1. Archival slides with or without microdissection

2. Lavage fluid from needles washing after direct

preparing direct smears

3. Cell block

4. Obtaining additional pass to be collected in preservative

solution

Specimen types for molecular testing

Specimen types

Comments

Scraping all cellular

Ability to select slides containing optimal numbers
materials from archival
of tumor cells
slides
Destruction of diagnostic materials on the original
slides
Microdissecting from
Labor intensive with microdissection
archival slides
Lavage fluid from
washing the needles
after preparing direct
smears

Quantity and composition of cellular materials

unknownCompromise sensitivity
Discordance of BRAF mutation status between
matched FNA and FFPE samples
Storage up to 3 weeks after collection

Obtaining additional
pass to be collected in
preservative soln

Quantity and composition of cellular materials

unknownCompromise sensitivity
Discordance of BRAF mutation status between
matched FNA and FFPE samples
Required different collection protocol

Conclusions & Future Directions

Molecular testing improved diagnostic accuracy for
thyroid FNA

High PPV for cancer except RAS

Need to define algorithms of patient management based
on cytology and molecular testing

Prognostic markers

BRAF mutation signifies more aggressive tumor
behaviordifferent therapeutic regime

Therapeutic targets

Novel inhibitors of MAP kinase pathway